Methods for determining amount of functional albumin

ABSTRACT

A method for determining an amount of functional albumin includes providing a test sample containing a defined amount of albumin of unknown binding capacity and a reference sample containing the same defined amount of albumin having a reference binding capacity, incubating the test and reference samples with a defined amount of at least one albumin-binding marker M under conditions that allow formation of complexes of the at least one albumin-binding marker M and albumin (M:A), removing the complexes, detecting a presence or an amount of unbound marker M in the samples after removal of the complex (M:A) through a first and a second test strips that allow for a determination of an amount of unbound marker M, and determining the amount of functional albumin based on the presence or the amount detected of marker M.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 16/607,064, filed Oct. 21, 2019, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/EP2018/060176, filed Apr. 20, 2018, designating the U.S. andpublished as WO 2018/193087 A1 on Oct. 25, 2018, which claims thebenefit of Deutsch Application No. DE 10 2017 206 786.1, filed Apr. 21,2017. Any and all applications for which a foreign or a domesticpriority is claimed is/are identified in the Application Data Sheetfiled herewith and is/are hereby incorporated by reference in theirentireties under 37 C.F.R. § 1.57.

FIELD

The present invention relates to methods for determining the relativebinding capacity of albumin The invention further relates to a methodfor determining the amount of functional albumin.

SUMMARY

The present invention relates to methods for determining the relativebinding capacity of albumin by means of test strips. In particular, theinvention relates to a method for determining the relative bindingcapacity of albumin that comprises the following steps: a) providing atleast two measurement solutions of a test sample and of a referencesample, wherein the measurement solutions contain at least onealbumin-binding marker M and this at least one albumin-binding marker Min at least one measurement solution of the test sample and of thereference sample exceeds the presumed available binding capacity ofalbumin and wherein the test sample contains a defined amount of albuminof unknown binding capacity and the reference sample contains the samedefined amount of albumin having a reference binding capacity; b)incubating the measurement solutions under conditions that allow the atleast one albumin-binding marker M to bind to albumin to form complexesof this marker M and albumin (M:A); c) removing the complexes (M:A)produced in step b); d) detecting the presence or amount of unboundmarker M in the solutions after removal of the complex (M:A) by at leastone test strip that allows determination of the unbound marker; and e)determining the relative binding capacity of albumin in the test samplebased on the presence or detected amounts of unbound marker M in stepd). The invention further relates to a method for determining the amountof functional albumin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Decrease in albumin function and increase in uremic toxin loadwith increasing severity of kidney failure

FIG. 2: Decrease in albumin function with increasing severity of liverfailure, illustrated on the basis of the CHILD and MELD clinicalclassification systems

FIGS. 3A and 3B: Determination of the relative binding capacity ofalbumin in plasma samples from patients with chronic liver damage(patients 4, 5, 6) and patients with end-stage renal insufficiency(patients 1, 2, 3), in a stabilizer-containing pharmaceutical albuminpreparation, and in plasma from a healthy volunteer. FIG. 3A) Diazepamconcentration [μmol/l] in the last sample in which free diazepam wasstill not detectable in the filtrate (cut-off 200 ng/ml=0.7 μmol/l).FIG. 3B) Relative albumin-binding function of binding site II (rABFII).

DETAILED DESCRIPTION

Albumin, which is the protein present in highest concentration in humanplasma, is responsible, in addition to other functions such as themaintenance of colloid osmotic pressure, for the transport of various,mostly lipophilic substances in the body. In addition to numerousendogenous substances, metabolic products, and hormones, for examplefatty acids, bilirubin, bile acids, indoxyl sulfate, tryptophane,steroids and cytokines, a large number of drugs are transported inalbumin-bound form in the body (Kragh et al., Practical aspects of theligand-binding and enzymatic properties of human serum albumin. BiolPharm Bull 2002 June; 25(6): 695-704.)

In addition to 7 binding sites for long-chain fatty acids and a free SHgroup on cysteine 34, for example for nitric oxide, there are twogroup-specific binding sites available for the diversity of endogenousor administered (exogenous) substances. With reference to the studies onthe characterization of albumin binding sites by Sudlow and coworkers,these are referred to as binding sites I and II (Sudlow G, et al., Thecharacterization of two specific drug binding sites on human serumalbumin, Mol Pharmacol 1975; 11(6): 824-832). Whereas it is mainlyheterocyclic substances or dicarboxylic acids that undergo binding atbinding site I, often also referred to as the warfarin/bilirubin bindingsite, the ligands of binding site II, often also referred to as thediazepam/indole binding site, mainly have an aromatic basic structure.

The binding of a substance to the albumin molecule can be influenced byinteractions with other albumin-bound substances that are competing forthe same binding site. In addition to these competitive displacementmechanisms, allosteric interactions or post-translational structuralalterations of the albumin structure, for example by carbamylation orglycation, can alter the binding to albumin of exogenous toxins andendogenous substrates (Lee P., Wu X., Review: Modifications of humanserum albumin and their binding effect. Curr Pharm Des 2015; 21(14):1862-1865 and Fassano M. et al., The extraordinary ligand bindingproperties of human serum albumin IUBMB Life. 2005 December; 57(12):787-96).

Under physiological conditions, the state of albumin loading is low. Inindividuals with impaired elimination function, for example due to liverand/or kidney failure, this can lead to an accumulation of lipophilicalbumin-bound substances in the blood and thus to greater saturation ofthe albumin binding sites or overloading of the albumin molecule. Thiscan result, through competitive or allosteric interactions, in anincrease in the active, unbound proportion of substrates, cytokines,hormones, and drugs and can be associated with influencing metabolicfeedback loops and altering pharmacological effects and also withincreased side effects.

Various substances found in elevated concentrations in the plasma orcerebrospinal fluid of patients with liver failure have been identifiedin recent years and their significance for the clinical course of liverfailure made clearer. Ammoniac, short- and medium-chain fatty acids,mercaptans, and phenols have long since been regarded as toxicsubstances in liver failure. Other substances such as bilirubin, bileacids or certain amino acids such as tryptophane, phenylalanine ortyrosine have been ascribed an at least indirect influence on metabolismvia displacement mechanisms on the albumin molecule or as precursors forphenols or inhibitory false neurotransmitters (Sen et al., Emergingindications for albumin dialysis. Am J Gastroenterol 2005 February;100(2): 468-475).

A number of different toxins, metabolites or cytokines are currentlyviewed as directly linked to complications of acute or chronic liverfailure, such as hepatic encephalopathy (HE), hyperdynamic circulation,portal hypertension, cholestasis, pruritus, ascites, and hepatorenalsyndrome (HRS). With increasing knowledge of the pathogeneticrelationships of these substances, there is increasing focus too on thequestion of their therapeutic elimination and consequent influence onthe severity, course or prognosis of liver failure (Hughes R D. Reviewof methods to remove protein-bound substances in liver failure. Int JArtif Organs 2002 October; 25(10): 911-917).

By eliminating albumin-bound substances in liver failure by means ofalbumin dialysis (usually in the form of the Molecular AdsorbentRecirculating System—MARS®), it was possible in randomized controlledstudies to avoid or reduce complications of liver failure such as renalfunction, mental function (hepatic encephalopathy) or hemodynamics andto significantly reduce mortality (Heemann et al. Albumin dialysis incirrhosis with superimposed acute liver injury: A prospective,controlled study. Hepatology 2002 October; 36(4): 949-958).

In kidney failure, inadequate elimination results in an accumulation ofvarious uremic toxins, including low-molecular-weight albumin-boundtoxins. Indoxyl sulfate and p-cresol sulfate are prototypes for theseuremic toxins and are examined in clinical and experimental studies.Clinical studies have shown that the rising concentrations ofalbumin-bound uremic toxins associated with increasing renalinsufficiency have toxic effects on the kidneys and vascular endothelium(Liabeuf et al., Protein-bound uremic toxins: new insight from clinicalstudies. Toxins (Basel) 2011 July; 3(7): 911-919). Indoxyl sulfate haspro-oxidative and pro-inflammatory properties and boosts expression ofpro-inflammatory cytokines such as TGF-β. Moreover, toxin-dependentfibrosis of renal tissue by indoxyl sulfate was detected both in a 5/6nephrectomy model and in a hypertensive rat model. Pro-inflammatoryeffects of p-cresol sulfate on leukocytes and an influence of indoxylsulfate on aortic calcification and arterial stiffness were alsodemonstrated.

A direct effect of endogenous uremic toxins on the proliferation andviability of cell cultures has been confirmed. This adverse effect islikely to be functional in nature, since it was not accompanied by anincrease in apoptosis (Dou et al. The uremic solutes p-cresol andindoxyl sulfate inhibit endothelial proliferation and wound repair.Kidney Int 2004 February; 65(2): 442-451). These experiments likewiseshowed albumin-bound uremic toxins to have an adverse effect on woundhealing.

Irrespective of whether the impaired albumin function in organ failureis caused by increased loading with albumin-bound substances, allostericinteractions or by oxidative structural changes to the albumin molecule,or by a combination of these factors, it results in altered binding ofendogenous substances or exogenously administered drugs and thus in anincrease in the unbound fraction. However, because it is only the freefraction of a substance that is pharmacologically active, this resultsin a stronger effect.

A high degree of loading would be indicative of elevated freeconcentrations of albumin ligands and thus of greater adverse toxiceffects. On the other hand, if the physiological binding function of thealbumin molecule is present or restored, effects of toxins would bereduced, the physiological plasma transport of substrates andmetabolites maintained, and the pharmacokinetic-pharmacodynamicrelationships to be expected in drug therapy observed.

One option for determining the available binding function of the albuminmolecule is to determine the individual concentration of all competingsubstances and their cumulative total and to estimate the degree ofmolar loading of the albumin molecule. However, the determination ofcertain ligands, for example the uremic toxins indoxyl sulfate andp-cresol sulfate, is possible only in a laborious technical process byHPLC. Even if the analysis of these two uremic toxins could besimplified to such a degree as to allow rapid analysis at the patient'sbedside, the absence of any determination of the concentrations of otherknown, and in particular hitherto unknown, toxins would mark thisapproach down as unpromising.

Another approach would be to determine the albumin load regardless ofthe nature and fractions of the substances occupying the binding site.If such an easily determinable parameter were available, it could beused in clinical studies and its suitability as a prognostic parametercould be examined in studies with different patient populations. Byanalogy, for example for the determination of blood lipids and if thedata situation permits this, it would then be possible to use it inroutine diagnostics to assess the individual prognosis, to select theappropriate patient-specific therapy, and to monitor the therapeuticoutcome and make any subsequent adjustments to the treatment plan.

Albumin function has been shown to correlate with severity of disease inboth liver and kidney failure (Klammt et al. Albumin-binding function isreduced in patients with decompensated cirrhosis and correlatesinversely with severity of liver disease assessed by model for end-stageliver disease. Eur J Gastroenterol Hepatol 2007 March; 19(3): 257-263.;Klammt et al. Albumin-binding capacity (ABiC) is reduced in patientswith chronic kidney disease along with an accumulation of protein-bounduraemic toxins. Nephrol Dial Transplant 2011 Nov. 15; 27(6): 2377-2383).In a randomized controlled clinical trial, the elimination ofalbumin-bound substances was associated with an improvement in albuminfunction and mortality. Patients who recorded an improvement in ABiC(albumin binding capacity) in the first week of treatment showedsignificantly higher survival than patients with no improvement inalbumin function (Klammt et al. Improvement of impaired albumin bindingcapacity in acute-on-chronic liver failure by albumin dialysis. LiverTranspl 2008 Aug. 28; 14(9): 1333-1339). In an initial pilot study,impaired albumin function was likewise demonstrated in the group ofseptic patients on the basis of the ABiC test, which correlates withseverity (SAPS II) (Hinz et al., Albumin function is reduced in severesepsis. Infection 2011; 39: S118).

EP 1 315 973 B1 discloses an indirect method for the quantitativedetermination of the existing binding capacity of albumin in an aqueoussolution. In a preferred embodiment, the marker substance dansylsarcosine is used and its unbound fraction determined by fluorescencespectrophotometry after binding to a defined albumin as fluorescenceenhancer.

However, the fluorometric determination of the binding capacity ofalbumin necessitates various steps with the result that thedetermination in the laboratory is currently performed some time aftersample collection. If simplifying the analysis could enabledetermination in the vicinity of the patient (point of care), this wouldallow individual treatment planning, for example the administration ofinfusions, dosage of drugs, start, duration, and intensity ofextracorporeal procedures, to take account of the patient's currentcondition and reduce both the risk of overtreatment that is associatedwith side effects and underdosing that is risky for the patient.

It is therefore the object underlying the present invention to provide arapid and simple method for determining the relative binding capacity ofalbumin that allows point-of-care diagnostics.

The object is achieved by the embodiments described in the claims andhereinafter.

The invention thus relates to a method for determining the relativebinding capacity of albumin that comprises:

a) providing at least two measurement solutions of a test sample and ofa reference sample, wherein the measurement solutions contain at leastone albumin-binding marker M and this at least one albumin-bindingmarker M in at least one measurement solution of the test sample and ofthe reference sample exceeds the presumed available binding capacity ofalbumin and wherein the test sample contains a defined amount of albuminof unknown binding capacity and the reference sample contains the samedefined amount of albumin having a reference binding capacity;

b) incubating the measurement solutions under conditions that allow theat least one albumin-binding marker M to bind to albumin to formcomplexes of this marker M and albumin (M:A);

c) removing the complexes (M:A) produced in step b);

d) detecting the presence or amount of unbound marker M in the solutionsafter removal of the complex (M:A) by at least one test strip thatallows determination of the unbound marker; and

e) determining the relative binding capacity of albumin in the testsample based on the presence or detected amounts of unbound marker M instep d).

In addition to the above steps, the present method may also includefurther steps. A further step may, for example, be the addition offurther substances, for example the addition of a substance to the testsample and reference sample to stabilize the albumin.

The method according to the present invention may preferably beautomated. This allows the treatment of the test and/or referencesample(s), for example the incubation of the measurement solutions andremoval of the complexes formed from this marker M and albumin (M:A)and/or detection by means of test strips and determination of therelative binding capacity of albumin, to be performed by suitablerobotic instruments, analysis robots and/or be computer-assisted.

For the purposes of the present invention, “sample” is understood asmeaning a solution containing albumin. It is preferably an aqueoussolution having a pH of between 5 and 8, particularly preferably havinga pH of between 7 and 8. The so-called “test sample” according to theinvention should contain a defined amount of albumin of unknown bindingcapacity, whereas the so-called “reference sample” should contain thesame defined amount of albumin having a reference binding capacity. Whatis to be understood by the term “binding capacity” is explained indetail elsewhere herein. A “defined amount” of albumin is understood asmeaning an approximately defined amount of albumin that is measurable.The amount of albumin is usually determined by determining theconcentration of albumin in a solution.

Methods and means for determining the albumin concentration, for examplethe amount of human serum albumin in the serum of a patient, are knownto those skilled in the art and include, for example, determination withbromocresol green, immunochemical methods including immunoturbidimetry,and (protein) electrophoresis. It is moreover known to those skilled inthe art that typically, in a human sample, between about 10 and 60 gramsof albumin can be detected in one liter of blood and that human serumpreferably contains about 35 to 55 grams of albumin per liter. It isadditionally known to those skilled in the art that the calculatedamount of albumin provides information on the number of albuminmolecules present, but not on their (binding) function. A largecalculated amount of albumin may, for example, exhibit low bindingfunctionality, i.e. a marker that binds specifically to albumin may nolonger bind to albumin despite the presence of albumin molecules becauseall the binding sites for this marker on the albumin molecules presentare already occupied. Conversely, a small calculated amount of albuminmay exhibit high binding functionality if, for example, certain or allof the binding sites of the albumin molecules in the solution are free.Thus, by contrast with the calculated amount of albumin, it is thefunctional amount of albumin, or the amount of albumin that is able totake up an albumin-binding marker M, that allows conclusions to be drawnabout the binding capacity of albumin, which is explained in detailelsewhere herein.

The “test sample” according to the invention is preferably a blood,serum or plasma sample of a patient. It is further preferable if thetest sample is an albumin-containing solution, i.e. an aqueous solutioncontaining albumin, more preferably a pharmaceutical albumin preparationor cell culture supernatants.

The term “patient” generally refers to a human subject being monitoredand/or treated for a medical condition, illness or similar by ahealthcare professional. For the purposes of the invention, the patientis preferably a human subject with liver damage and/or renalinsufficiency and/or sepsis. The patient is further preferably someonewho is to receive or has received a plurality of albumin-bound drugs orsubstances that can bind to albumin. The terms liver damage, renalinsufficiency, and sepsis include here all acute and chronic diseasestates. The patient preferably has chronic liver damage and/or chronicrenal insufficiency and/or severe sepsis with secondary organ damage.Symptoms and characteristics of the abovementioned conditions are knownto those skilled in the art and are described, for example, in standardmedical textbooks such as Stedman or Pschyrembl. However, patients forthe purposes of the invention may also be animals, for example mammalsand in particular domestic and farm animals such as dogs, cats, horses,cows, pigs or sheep, or laboratory animals such as mice or rats.

The “reference sample” according to the invention is preferably asynthetically produced albumin solution or a sample from a healthysubject of the same species, also referred to hereinafter as a“subject”. The term “synthetically produced albumin solution” covers anytype of aqueous solution to which albumin has been added and/or anaqueous solution known to contain albumin. A synthetically producedalbumin solution covers, for example, albumin-containing solutions suchas pharmaceutical albumin preparations. For example, a syntheticallyproduced albumin solution may be phosphate-buffered saline (PBS) towhich a defined amount of albumin has been added. Albumin-containingsolutions that are pharmaceutically produced and/or commerciallyavailable as medicinal products may also serve as a reference solution.According to the invention, the reference sample is particularlypreferably a sample from a healthy subject, a pooled sample from severalsubjects, a pharmaceutical albumin-containing preparation or asynthetically produced albumin solution.

A “healthy subject” is preferably a human who is apparently healthy andis known to have no liver damage, no renal insufficiency, and no sepsis,and thus no elevated saturation of albumin binding sites or overloadingof the albumin molecule. The sample from a healthy subject may, for thepurposes of the invention, moreover be a “pooled” sample, i.e. a mixturefrom a number of healthy subjects. These are preferably pooled plasmadonations from a number of healthy subjects, which may be available, forexample, via blood banks. The reference sample may be largely identicalto the test sample, differing only in the binding capacity of albumin,as explained in detail elsewhere herein.

The term “measurement solution” for the purposes of the presentinvention refers to a solution that comprises a part-volume of the testsample or of the reference sample and that contains at least onealbumin-binding marker M. What is to be understood by the term“albumin-binding marker M” is explained in detail elsewhere herein. Ameasurement solution of the test sample may, for example, be an aliquotof the test sample or a diluted solution of the test sample to which theat least one albumin-binding marker M is added. According to theinvention, at least two measurement solutions of the test sample and ofthe reference sample are to be provided, wherein the albumin-bindingmarker M exceeds the presumed available binding capacity of albumin (asexplained elsewhere herein) in at least one measurement solution of thetest sample and of the reference sample. The measurement solution shouldpreferably have a pH of between 5 and 8, particularly preferably between7 and 8. The measurement solution may also contain additionalsubstances, for example buffers and/or stabilizers used to stabilize thepH and/or to stabilize the albumin. It is known to those skilled in theart that the albumin concentration in the serum of a healthy subject isabout 35 to 55 grams per liter, which means that a part-volume, forexample a diluted solution of the serum, should have an albuminconcentration of less than 55 grams per liter. It is also known to thoseskilled in the art that different measurement solutions having varyingmolar ratios of marker M and albumin may be produced through dilutionseries of the test sample and of the reference sample, on addingconstant amounts of marker M, or by dividing the test sample and thereference sample into aliquots and adding varying amounts of marker M.Preference is given to molar ratios (marker M/albumin) of, for example,1, 0.5, 0.3, 0.25, 0.2, 0.15, 0.1, and 0.05. According to the invention,the marker M should exceed the presumed available binding capacity ofalbumin in at least one measurement solution of the test sample and ofthe reference sample, as explained elsewhere herein.

According to the invention, at least two measurement solutions of thetest sample and of the reference sample need to be provided. Theprovision of at least two measurement solutions means that preferablytwo measurement solutions of the test sample and two measurementsolutions of the reference sample are provided. Step a) of the methodaccording to the invention thus comprises preferably the provision of atleast two measurement solutions each of a test sample and of a referencesample. It is further preferable to provide at least 3, 4, 5, 6, 7 or 8measurement solutions of the test sample and of the reference sample.For example, the test sample and the reference sample may each be splitinto 6 aliquots that contain the same defined amount of albumin. This isfollowed by addition of different amounts of an albumin-binding marker Mto give decreasing molar ratios of marker to albumin in the respectivealiquots of the test sample and of the reference sample. Alternatively,increasing dilutions of the test sample and of the reference sample maybe prepared, for example 6 dilutions of the test sample and of thereference sample, each containing different amounts of albumin, may beprepared. This is followed by the addition of equal amounts of analbumin-binding marker M so that varying molar ratios of marker toalbumin are in turn present in the respective 6 dilutions of the testsample and of the reference sample. According to the invention, in atleast one measurement solution of the test sample and of the referencesample here, the at least one albumin-binding marker M needs to exceedthe presumed available binding capacity of albumin, i.e. so that themarker is present in “excess” relative to the binding capacity ofalbumin. Incubation of the at least 2, preferably also 3, 4, 5, 6, 7 or8 measurement solutions results in the albumin-binding marker M beingable to bind to at least one defined albumin binding site and theformation of complexes from the marker and albumin (M:A). After removalof the complexes (M:A) formed, the amount of unbound marker M may bedetected through the use of at least one test strip. This detection mustbe possible in at least one of the measurement solutions of the testsample and of the reference sample. It is accordingly then possible, forexample, to determine which of the at least 2, preferably 3, 4, 5, 6, 7or 8 measurement solutions of the reference sample is the measurementsolution in which unbound marker M is first detectable using at leastone test strip and to determine which of the at least 2, preferably 3,4, 5, 6, 7 or 8 measurement solutions of the test sample is themeasurement solution in which no unbound marker M is last detectableusing at least one test strip. What exactly is to be understood by theterm “test strip” is explained in detail elsewhere herein. Based on thedetected amounts of unbound marker M, it is then possible to determinethe relative binding capacity of albumin, also referred to as therelative binding function of albumin, which provides information on thebinding site-specific loading state and on the remaining availablebinding capacity of albumin at the defined binding site(s), as alsoexplained in more detail elsewhere herein.

For the purposes of the invention, the term “binding capacity ofalbumin” is understood as meaning the capacity of the albumin to takeup/bind one or more substances that bind specifically to albumin. Theterm binding capacity covers the uptake capacity of one or moresubstances at one and/or more binding sites. When the binding capacityat a particular binding site, for example binding site I and/or II, isreached, this may also be referred to as “binding site I and/or IIsaturation”. For example, if a substance such as diazepam, which bindsspecifically to binding site II, completely blocks binding site II,binding site II is saturated and excess diazepam still present in thesolution is no longer able to bind to albumin. The formation ofcomplexes of albumin and diazepam in said solution (with albuminsaturated at binding site II) is accordingly no longer possible and anydiazepam present remains unbound in the solution. The binding capacityof albumin is therefore to be regarded as a functional property ofalbumin. As previously mentioned above, a large amount or highconcentration of albumin may, for example, exhibit low bindingfunctionality (“bad albumin” for an albumin-binding marker), whereas asmall amount or low concentration of albumin may exhibit high bindingfunctionality (“good albumin” for an albumin-binding marker).

The term “reference binding capacity” refers to an assumed or knownbinding capacity. This may, for example, be the binding capacity of asynthetic solution to which albumin has been added and of which it isknown that the binding sites of the albumin are unoccupied. It canadditionally be a sample, preferably a serum or plasma sample, from ahealthy subject, preferably a subject without liver damage or renalinsufficiency, in whom pronounced saturation of the albumin bindingsites or overload of the albumin molecule are known to be absent. Thus,in comparison to an unwell patient, preferably a patient with liverdamage and/or renal insufficiency, the binding capacity of albumin inthe sample from the healthy subject (i.e. the reference sample) will beknown or assumed to be higher than would be the case in said patient(i.e. in the test sample).

The “relative binding capacity of albumin”, also referred to as the“relative binding function of albumin”, which is determined by themethod according to the invention, provides information on the bindingsite-specific loading state and on the remaining available bindingcapacity of albumin at said binding site(s). According to the invention,a substance that can bind to albumin is also referred to as an albuminligand or albumin-binding marker, as described elsewhere herein.

The term “exceeding the binding capacity” of albumin means that analbumin-binding marker M is no longer able to bind to albumin and thusexceeds the binding capacity of albumin. For example, if more marker Mis added to a solution containing albumin than is able to bindspecifically to the albumin present in the solution, the marker exceedsthe binding capacity of albumin. Unbound marker M that is not bound toalbumin/is not present in marker:albumin (M:A) complexes will then bedetectable in the solution (preferably after removal of marker:albumincomplexes). For example, if binding site II is completely occupied by aspecific marker such as diazepam, i.e. binding site II is saturated, theaddition or presence of diazepam that is now no longer able to bind toalbumin (because the binding site is saturated) results in the bindingcapacity at said binding site II being exceeded. However, in a solutioncontaining albumin in which binding site II is already saturated, anyfurther markers that, for example, bind specifically to binding site Imay continue to be taken up by the albumin present in the solution untilbinding site I is likewise saturated. The presumed available bindingcapacity or an exceeding of this binding capacity is thus dependent onthe pre-existing loading state of the albumin molecule and theavailability of binding site(s) to which the albumin-binding marker(s)bind. Moreover, it is known to those skilled in the art that, even ifthe binding capacity of the individual binding sites is altered byallosteric interactions or by structural changes in the albuminmolecule, account will likewise be taken of these changes in bindingproperties. As already mentioned above, it can be assumed that thepresumed available binding capacity of albumin in a sample from apatient with liver damage and/or renal insufficiency will be lower thanin a sample from a healthy subject, since liver damage and/or renalinsufficiency are known to be able to result in increased saturation oralbumin overloading.

What is more, exceeding the binding capacity of albumin for analbumin-binding marker M should according to the invention result in thepresence of “excess”/unbound marker M in the sample, i.e. unbound markerM in at least one measurement solution of the test sample and of thereference sample, being detectable after removal of the marker:albumincomplexes (as described in detail elsewhere herein). It is preferable ifthe excess/unbound marker M, particularly preferably the unbound markerdiazepam, is present in a concentration of at least 100 ng/ml, at least200 ng/ml, at least 300 ng/ml, at least 400 ng/ml or at least 500 ng/ml.

The term “albumin-binding marker M” is according to the inventionunderstood as meaning a substance that can bind specifically to albumin.It is possible here for the albumin-binding marker M to bind to one ormore binding sites in the albumin. According to the invention, thealbumin-binding marker M binds preferentially to binding site I and/orII of albumin. This includes, for example, substances known to thoseskilled in the art that have been shown to be able to bind to therespective binding sites of albumin, for example diazepam, otherbenzodiazepines, tryptophane, bile acids, dansyl sarcosine, medium-chainfatty acids, warfarin, furosemide, sulfonylureas, and dansyl amide, oreven opioids such as fentanyl, synthetic drugs such as cocaine, orcannabinoids.

For the purposes of the invention, the albumin-binding marker M ispreferably a benzodiazepine, particularly preferably diazepam. Themethod according to the invention is to include at least onealbumin-binding marker M. The use of multiple markers is, however, alsopreferred, for example it is possible to use a combination of markersthat bind to different binding sites in the albumin molecule or acombination of markers that bind to the same binding site, for exampleto binding site I of albumin. Markers that bind to the same binding sitemay in turn be substances that bind with equal, similar or differentbinding strength to defined binding site(s), for example binding site Iof albumin.

The term “binding site I”/“binding site II” of albumin refers to twobinding sites of albumin to which substances can specifically bind. Itis known to those skilled in the art that, in addition to 7 bindingsites for long-chain fatty acids and a free SH group on cysteine 34, fornitric oxide for example, albumin has two group-specific binding sitesavailable for the diversity of endogenous or administered (exogenous)substances. With reference to the studies on the characterization of thealbumin binding sites by Sudlow and co-workers, these are referred to asbinding sites I and II (Sudlow G, et al., The characterization of twospecific drug binding sites on human serum albumin Mol Pharmacol 1975;11(6): 824-832). Those skilled in the art will moreover be aware thatbinding site I of albumin is often referred to as the warfarin/bilirubinbinding site and binds mainly heterocyclic substances or dicarboxylicacids, whereas binding site II is often also referred to as thediazepam/indole binding site and binds mainly ligands having an aromaticbasic structure. For example, warfarin, furosemide or dansyl amide bindpreferentially to binding site I, whereas indoles such as tryptophane orelse diazepam, bile acids, dansyl sarcosine and medium-chain fatty acidsbind preferentially to binding site II (Peters T. All about albumin:biochemistry, genetics, and medical applications. Academic Press, 1996;Ghuman J et al., Structural basis of the drug-binding specificity ofhuman serum albumin. J Mol Biol (2005); 353: 38-52).

According to the invention, the incubation of the measurement solutionsis to be carried out under conditions that allow the at least onealbumin-binding marker M to bind to albumin to form complexes of thismarker M and albumin (M:A). These are preferably complexes of abenzodiazepine, preferably diazepam, and albumin. The incubation of themeasurement solutions should preferably take place at room temperaturefor up to 30 minutes, particularly preferably for at least 10 seconds,20 seconds, 45 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15minutes, 20 minutes or 30 minutes.

The term “removal of the complexes” is understood as meaning theseparation of the complexes formed from at least one albumin-bindingmarker M and albumin (M:A complexes) from the measurement solution.Methods and means of removing M:A complexes, preferably diazepam-albumincomplexes, are known to those skilled in the art and include, forexample, centrifugation, filtration or specific adsorption methods suchas immunoadsorption. Removal of the M:A complexes leaves a solution, forexample an albumin-free filtrate, in which M:A complexes are no longerpresent. The presence and/or amount of unbound marker M can now bedetermined in this solution, respectively.

The term “detection” as used herein includes the qualitative,semiquantitative, and/or quantitative determination of the presence ofunbound marker M in a solution. The term “detecting the presence oramount of unbound marker M” is understood as meaning the detection ofunbound marker M in at least one measurement solution of the test sampleor of the reference sample after removal of the M:A complexes (asexplained above). The term amount should be understood here not in thesense of an absolute amount, but rather as a semiquantitative statement,for example in the sense of a minimum amount above which the presence ofthe unbound marker is possible based on the detection limit of the teststrip used. Detecting the presence or amount of unbound marker Mconsists preferably of determining the qualitative presence of unboundmarker, i.e. a yes or no statement as to whether unbound marker ispresent in the solution into which the test strip is dipped.

The term “detection limit”, also referred to as the limit of detection,is understood as meaning the extreme value of a measurement method downto which the measured variable can still be reliably detected. Forexample, if a test strip for diazepam with a predetermined detectionlimit of 100 ng/ml is used, a corresponding “positive” signal for thetest strip means it is possible to conclude that unbound marker ispresent in said solution in a content of at least 100 ng/ml or, if acorresponding “negative” signal is obtained for the test strip, it canbe concluded that unbound marker is absent or is present in an amountthat is too small to be detected. It is known to those skilled in theart that the corresponding signals, which are to be regarded as eitherpositive or negative, depend on the substance to be detected and/or teststrip used and/or derive from the detection method of the test strip.The predetermined detection limit is according to the inventionpreferably at least 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml,300 ng/ml, 400 ng/ml or 500 ng/ml.

The term “tolerance limit”, also referred to as “cut-off”, is understoodas meaning the value or limit below/above which below/above which a testresult is to be evaluated as positive or negative. To ensure reliabilityof detection in a test or test strip and thus to avoid “false positive”results, the tolerance limit (cut-off) is usually several times higherthan the detection limit. The tolerance limit may, however, also be thesame as the detection limit. In a strip test, a so-called “positivesignal” is usually obtained when the marker concentration, for examplethe amount of unbound diazepam in the solution after removal of the M:Acomplexes, is below the specified tolerance limit (for example a cut-offvalue of 200 ng/ml). Preferred tolerance limits for test stripsaccording to the invention, preferably for a benzodiazepine,particularly preferably for diazepam, are about 100 ng/ml, 200 ng/ml,300 ng/ml, 400 ng/ml, 500 ng/ml, and 600 ng/ml.

It is also known to those skilled in the art that there are varyingpreferred tolerance limits for individual substances or test strips. Forexample, the tolerance limits may depend on the substance to be detectedand/or the test strip used and/or the detection method of the teststrip. In addition, there may also be pertinent regulatory aspects, forexample guidelines and requirements of the US National Institute on DrugAbuse (NIDA) for the approval of test strips for the detection of drugssuch as cocaine or amphetamine. This is to ensure that the decision,i.e. the threshold above which a test result is to be evaluated aspositive or negative, meets specific requirements. For example, a teststrip for opiates needs to ensure that the consumption merely of apoppyseed bun does not give a positive signal and that the presence ofopiates is judged to be positive only above a certain amount. Forstandard drug tests and corresponding test strips that are approved bythe NIDA, the tolerance limit is often much higher, e.g. hundreds orthousands of times higher, than the detection limit of the substance tobe detected. Test strips with different tolerance limits (cut-offs) fordifferent substances are known to those skilled in the art. For example,tolerance limits are preferably about 300 ng/ml, 500 ng/ml, 1000 ng/mlor 3000 ng/ml for the detection of amphetamine, about 100 ng/ml, 150ng/ml, 300 ng/ml or 400 ng/ml for cocaine, about 20 ng/ml or 100 ng/mlfor fentanyl, about 300 ng/ml, 1000 ng/ml or 2000 ng/ml for opiates suchas morphine, about 20 ng/ml, 50 ng/ml, 150 ng/ml or 500 ng/ml formarijuana (THC), about 100 ng/ml or 200 ng/ml for oxycodone, about 25ng/ml or 50 ng/ml for phencyclidine, and about 300 ng/ml, 500 ng/ml or1000 ng/ml for methamphetamine.

The term “test strip” covers any test strips or strip tests that can beused to detect a specific marker, preferably an albumin-binding markerM. Preference is given to test strips for use in aqueous solutions,preferably having a pH of between 5 and 8, particularly preferablyhaving a pH of between 7 and 8. Test strips for detectingbenzodiazepines are known to those skilled in the art. In addition, itis known to those skilled in the art that—depending on the test stripused—the presence or absence of a signal may mean a positive result. Forexample, commercially available test strips for benzodiazepines oftenemploy a competitive immunoassay. Detection in this case does not use asecond, labeled antibody, but a labeled competitor antigen (a syntheticcompound that is structurally similar to the analyte, for examplediazepam, and also binds to the antibody). This means there iscompetition between analyte (diazepam) and competitor for a binding siteon the antibody. The signal here behaves inversely to the analyteconcentration, i.e. little analyte=almost all antibody binding sites areoccupied by labeled competitor=>intense color reaction; muchanalyte=>weak color reaction. For the purposes of the invention, it ispreferably a test strip that is based on an immunochemical test forrapid detection of an albumin-binding marker having a visually readableresult. For example, a test strip based on a competitive sandwich ELISAthat allows the detection of benzodiazepines with a cut-off of 300 ng/mlby means of a visually readable control line and test line. If abenzodiazepine is present in the solution here, a colored line appears(control line). If no benzodiazepine is present in the solution, twocolored lines appear (test and control line).

According to the invention, the at least one test strip is intended toallow the determination of unbound marker M in the solutions afterremoval of the complexes of the marker and albumin (M:A) as describedelsewhere herein. Preference is moreover given to the use of more thanone test strip, preferably 3, 4, 5 or 6 test strips, having differentpredetermined detection limits and/or different predetermined tolerancelimits for one or more albumin-binding marker(s) M.

As already mentioned above, in most test strips a so-called “positivesignal” is obtained when the marker concentration, for example theamount of unbound diazepam in the solution after removal of the M:Acomplexes, is below the specified tolerance limit, for example a cut-offvalue of 200 ng/ml. For example, if two test strips are then used insaid solution, with the first test strip having a cut-off of 200 ng/mland the second a cut-off of 300 ng/ml, this allows direct,semiquantitative estimation of the free diazepam concentration in thesolution. In this example, the free diazepam concentration isaccordingly less than 200 ng/ml if a signal is generated in both teststrips used; if neither test strip gives a detectable test signal, thediazepam concentration in the solution is above 300 ng/ml. However, if asignal is observed on the test strip with a 300 ng/ml cut-off, but noton the test strip with the 200 ng/ml cut-off, the diazepam concentrationin the solution can be reported as being in the range between 200 and300 ng/ml. Thus, through suitable selection and combinations of teststrips, semiquantitative estimation of the diazepam concentration insaid solution is possible, thereby allowing determination of therelative albumin-binding function.

According to the invention, preference is given to the use of teststrips that allow the detection of benzodiazepines, particularlypreferably diazepam, or of synthetic drugs such as amphetamines,cannabis, methadone or opiates. Further preference is given to teststrips having different predetermined detection limits and/or differentpredetermined tolerance limits for one or more albumin-binding marker(s)M that are combined on a common carrier.

The “relative binding capacity of albumin” can also be referred to asthe relative albumin-binding function (rABFx) or “ABiC” (albumin bindingcapacity). The determination of the relative binding capacity of albuminis based, according to the invention, on the amounts of unbound markerin the solutions detected by means of test strips after removal of themarker:albumin (M:A) complexes.

The relative binding capacity of albumin (rABFx) is preferablydetermined according to the following formula:

${r\; A\; B\; F\; x} = \frac{{mD}_{S}}{{mD}_{R}}$

where x serves as the identifier of the specific binding site (forexample binding site I or II) and mDR and mDS represent the measurementsolution/molar dilution or ratio of marker to albumin (mD) at whichrespectively, by means of test strips according to the invention,unbound marker is first detected in the reference (mDR) and no unboundmarker is last detected in the sample (mDS). If the molardilution/measurement solution at which unbound marker is first detectedin the filtrate is lower in the sample than in the reference, therelative binding capacity of albumin is less than 1. If the two molardilutions are equal, the relative binding capacity of albumin is 1.

Compared to the known methods of the prior art, the method according tothe invention is fast, efficient, inexpensive, and can be used withoutany special infrastructure requirements. The method according to theinvention makes determination of determination of the relative bindingcapacity of albumin in the vicinity of the patient, i.e. point-of-carediagnostics, possible. This means that account can be taken of thepatient's current condition when planning treatment, for example theadministration of infusions, dosage of drugs, and start, duration, andintensity of extracorporeal procedures may be adjusted accordingly,thereby reducing side effects of overtreatment or the risk ofunderdosing the patient.

The definitions and explanations of terms provided previously likewiseapply to the embodiments described hereinafter.

In a preferred embodiment of the method according to the invention, theat least one albumin-binding marker M binds to binding site I and/or IIof albumin.

In another preferred embodiment of the method according to theinvention, the at least one albumin-binding marker M is abenzodiazepine. It is particularly preferable if the at least onealbumin-binding marker M is diazepam.

In a further preferred embodiment of the method according to theinvention, the at least one test strip has a predetermined detectionlimit and/or a predetermined tolerance limit for the at least onealbumin-binding marker M.

In a further preferred embodiment of the method according to theinvention, a plurality of test strips is used for differentalbumin-binding markers M.

In a further preferred embodiment of the method according to theinvention, the defined detection limit for the albumin-binding marker Mis at least 100 ng/ml and/or a predetermined tolerance limit is about200 ng/ml.

In a further preferred embodiment of the method according to theinvention, the test sample is a sample from a patient with liver damageand/or renal insufficiency and/or sepsis or an albumin-containingsolution.

In a further preferred embodiment of the method according to theinvention, the reference sample is a sample from a healthy subject or asynthetically produced albumin solution.

The invention further relates to a method for determining the amount offunctional albumin, comprising:

a) providing a test sample containing a defined amount of albumin ofunknown binding capacity and a reference sample containing the samedefined amount of albumin having a reference binding capacity;

b) incubating the test sample and reference sample with a defined amountof at least one albumin-binding marker M to the test sample and to thereference sample under conditions that allow the at least onealbumin-binding marker M to bind to albumin to form complexes of thismarker M and albumin (M:A);

c) removing the complexes (M:A) formed in step c);

d) detecting the amount of unbound marker M in the samples after removalof the complex (M:A) through a first and a second test strip that allowdetermination of the amount of unbound marker, with the test stripshaving different predetermined tolerance limits; and

e) determining the amount of functional albumin based on the detectedamounts of marker M in step d).

The test sample and the reference sample are for the purposes of theinvention a solution containing albumin, as explained in detailelsewhere herein. This is, moreover, an in vitro method. The provisionof a sample does not according to the invention comprise a method thatis carried out on the human body.

In a preferred embodiment of the method according to the invention, thefirst test strip has a predetermined tolerance limit, particularlypreferably for diazepam, of about 200 ng/ml and/or the second test striphas a predetermined tolerance limit of about 300 ng/ml.

It is further preferable if a tolerance limit for the detection of anamphetamine is about 300 ng/ml for the first test strip and about 500ng/ml for the second test strip,

a tolerance limit for the detection of cocaine is about 100 ng/ml forthe first test strip and about 300 ng/ml for the second test strip, atolerance limit for the detection of fentanyl is about 20 ng/ml for thefirst test strip and about 100 ng/ml for the second test strip, atolerance limit for the detection of an opiate such as morphine is about300 ng/ml for the first test strip and about 1000 ng/ml for the secondtest strip, a tolerance limit for the detection of marijuana (THC) isabout 20 ng/ml for the first test strip and about 150 ng/ml for thesecond test strip, a tolerance limit for the detection of oxycodone isabout 100 ng/ml for the first test strip and about 200 ng/ml for thesecond test strip, a tolerance limit for the detection of phencyclidineis about 25 ng/ml for the first test strip and about 50 ng/ml for thesecond test strip, a tolerance limit for the detection ofmethamphetamine is about 300 ng/ml for the first test strip and about500 ng/ml for the second test strip.

Preference is moreover given to the use of more than one test strip,preferably 3, 4, 5 or 6 test strips having different predetermineddetection limits and/or different predetermined tolerance limits for oneor more albumin-binding marker(s) M. According to the invention,preference is given to the use of test strips that allow the detectionof benzodiazepines, particularly preferably diazepam, or of syntheticdrugs such as amphetamines, cannabis, methadone or opiates. Furtherpreference is given to test strips having different predetermineddetection limits and/or different predetermined tolerance limits for oneor more albumin-binding marker(s) that are combined on a common carrier.

According to the invention, the amount of unbound marker M in thesamples after removal of the complex (M:A) is determined by means of atleast two test strips having different tolerance limits. Furtherpreference is given to the use of more than two test strips, preferably3, 4, 5, 6, 7 or 8 test strips having different tolerance limits and/orcombinations of a plurality of strip tests having different tolerancelimits (cut-off values).

For example, by using a plurality of test strips specific for marker M(preferably diazepam) having different cut-off values such as 100 ng/ml,200 ng/ml, 300 ng/ml, 400 ng/ml, and 500 ng/ml that are combined on acarrier, it is possible to determine the amount of unbound marker M in asample and for a reference solution (both of approximately the samealbumin concentration and containing approximately the same amount ofalbumin-binding marker M) and to deduce from this the functional albuminfraction and the albumin-binding function.

The determination of the relative binding capacity and the calculationof the relative albumin-binding function (rABFx) can preferably bedetermined according to the following formula:

${r\; A\; B\; F\; x} = \frac{C_{R}}{C_{S}}$

where x serves as the identifier of the specific binding site (forexample I or II) and CS or CR as the amount/concentration of the unboundmarker in the sample (CS) and reference (CR) after step d) of the methodaccording to the invention, for example the diazepam concentration inthe filtrate after removal of the marker albumin:marker (M:A) complexesin the test sample and reference sample according to the invention.

In a further preferred embodiment of the method according to theinvention, the at least one albumin-binding marker M binds to bindingsite I and/or II of albumin.

In another preferred embodiment of the method according to theinvention, the at least one albumin-binding marker M is abenzodiazepine. It is particularly preferable if the at least onealbumin-binding marker M is diazepam.

In a further preferred embodiment of the method according to theinvention, the test sample is a sample from a patient with liver damageand/or renal insufficiency and/or sepsis or an albumin-containingsolution.

In a further preferred embodiment of the method according to theinvention, the reference sample is a sample from a healthy subject or asynthetically produced albumin solution.

The invention further covers a method in which the detection of thepresence/amount of unbound marker M may be performed directly in thetest sample and the reference sample without having to perform aseparation step and/or removal of albumin:marker (M:A) complexes,respectively. This is achieved by using at least one test strip thatreacts specifically with the unbound marker M. For example, the teststrip(s) may be encased in a synthetic or biological membrane having adefined pore size that allows only the unbound marker molecules to comeinto direct contact with the test strip, whereas the albumin-boundmarker molecules are held back because of the size of the albuminmolecule and thus cannot be detected.

The content of all references cited herein is hereby incorporated byreference to the content of the relevant specific disclosures and in itsentirety.

EXAMPLES

The following examples are provided as illustration of the invention.They should not be construed in a restrictive manner with regard to thescope of protection.

Example 1: Principle of Determining the Relative Albumin Function (rABF)or Relative Binding Capacity of Albumin

To an albumin-containing sample S is added a specific marker M thatbinds to the albumin molecule and the amount of unbound marker MS isquantified by means of a strip test. In parallel, the same amount of thespecific marker M is added to an albumin-containing reference solution Rhaving the same albumin concentration as the sample S and the amount ofunbound marker MR in the reference solution is likewise detected.

Example 2: Detecting the Presence of Unbound Markers by Means of aMarker-Specific Strip Test

To multiple aliquots of a sample P and of a reference R, all having thesame albumin concentration, are added different amounts of marker so asto obtain a series of descending marker/albumin molar ratios for thesample and for the reference (for example 0.3, 0.25, 0.2, 0.15, 0.1, and0.05). Following a separation step, the presence of the unbound markerin the albumin-free filtrates of the individual molar ratios for thesample and for the reference is analyzed by means of a marker-specifictest strip having a defined cut-off (for example 200 ng/ml fordiazepam). The relative albumin-binding function (rABF) is determinedaccording to the following formula:

${r\; A\; B\; F\; x} = \frac{{mD}_{S}}{{mD}_{R}}$

where x serves as the identifier of the specific binding site (forexample I or II) and mDR and mDS are the molar dilutions at whichrespectively, in the filtrate by means of the strip test, unbound markeris first detected in the reference and no unbound marker is lastdetected in the sample.

If the molar concentration at which unbound marker is first detected inthe filtrate is lower in the sample than in the reference, the relativealbumin-binding function has a value of less than 1. If the two molardilutions are equal, the relative albumin-binding function is 1.

Example 3: Method for Determining the Amount of FunctionalAlbumin/Detecting the Unbound Amount of Marker by Means of Combinationsof Strip Tests with Different Cut-Off Values

To a sample S and to a reference R, both having the same albuminconcentration, is added the amount of marker M and, after a separationstep, the concentration of the unbound marker in the albumin-freefiltrate of the reference and of the sample is determined usingmarker-specific test strips with different cut-off values (for example100, 200, 300, 400, 500 ng/ml for diazepam) and the relativealbumin-binding function (rABF) is determined according to the followingformula:

${r\; A\; B\; F\; x} = \frac{C_{R}}{C_{S}}$

where x serves as the identifier of the specific binding site (forexample I or II) and C_(R) and C_(S) as the diazepam concentration inthe filtrate of the sample and reference respectively.

Detection of the amount of unbound marker by means of a marker-specificstrip test could also take place in the sample P and in the reference(without a separation step), provided it can be ensured that only theamount of unbound marker can be detected by the strip test. This could,for example, be achieved by encasing the test strip(s) in a membrane(synthetic or biological) having a defined pore size that allows onlythe unbound marker molecules to come into direct contact with the teststrip, while the albumin-bound marker molecules are held back because ofthe size of the albumin molecule and cannot be detected.

Example 4: Determining the Relative Binding Capacity of Albumin inPlasma Samples from Patients with Chronic Liver Damage and Patients withEnd-Stage Renal Insufficiency

A plasma sample is divided into aliquots and PBS is added so as toobtain 8 aliquots with a volume of 0.9 ml and an albumin concentrationof 83.3 μmol/l. To each of these aliquots is added 0.1 ml of diazepamsolutions of different diazepam concentration so as to obtain 6 aliquotswith an albumin concentration of 75 μmol/l and diazepam concentrationsof 18, 15, 12, 9, 6, and 3 μmol/l (corresponding to a molar ratio ofdiazepam to albumin of 0.24, 0.2, 0.16, 0.12, 0.08, and 0.04).

After an incubation period, the unbound amounts of marker are separatedby centrifugation (Centrisart Sartorius, cut-off 20000 daltons). Theamount of diazepam in the filtrate is then determined by means of astrip test (test strip cut-off 200 ng/ml). In the strip test used, asignal is obtained if the diazepam concentration in the liquid(filtrate) is below the specified cut-off value (200 ng/ml). The sample(or diazepam concentration) is determined at which the signal is lastdetectable, i.e. the last concentration in the filtrate that was belowthe cut-off value. The same is done using a reference sample (e.g.healthy control) and the diazepam concentration at which the signal ofthe test strip was last observed in the ultrafiltrate is determined heretoo. The ratio of these two concentrations is calculated and therelative albumin-binding function of binding site II (rABF II) isdetermined according to the formula below.

${r\; A\; B\; F\mspace{14mu} I\; I} = {\frac{M_{R}}{M_{S}}\mspace{14mu}{or}\mspace{14mu}\frac{C_{M\; S}}{C_{M\; R}}}$

where M_(R) and M_(S) are the amount of unbound marker (if determiningthe concentrations) or C_(MS) and C_(MR) are the concentration/dilutionof the added marker at which respectively, in the filtrate by means ofthe strip test, unbound marker is first detected in the reference and NOunbound marker is last detected in the sample.

This was done using plasma from a healthy volunteer (reference), astabilizer-containing pharmaceutical albumin preparation, and 3 plasmasamples from patients with chronic liver damage and 3 patients withend-stage renal insufficiency (see FIGS. 3A/3B).

Example 5: Semiquantitative Estimation of the Diazepam Concentration byMeans of Test Strips having Different Detection Limits

A plasma sample is diluted with PBS so that the albumin concentration inthe sample is then 677 μmol/l. To 1.8 ml of this sample is added 0.2 mlof a diazepam solution having a concentration of 500 μmol/l so that thesample then has an albumin concentration of 600 μmol/l and a diazepamconcentration of 50 μmol/l, which corresponds to a diazepam/albuminmolar ratio of 0.083.

After an incubation period, the unbound amounts of marker are separatedby centrifugation (Centrisart Sartorius, cut-off 20000 daltons). Thediazepam concentration in the filtrate is then determined by means of(at least) two different strip tests (test strip cut-off 200 ng/ml and300 ng/ml). In the strip test used, a signal is obtained if the diazepamconcentration in the liquid (filtrate) is below the specified cut-offvalue (200 or 300 ng/ml). Estimation of the free diazepam concentrationin the filtrate is thus possible. In our working example, the freediazepam concentration accordingly is less than 200 ng/ml if a signal isgenerated in both test strips used; if neither test strip gives adetectable test signal, the diazepam concentration in the filtrate isabove 300 ng/ml.

However, if a signal is observed on the test strip with a 300 ng/mlcut-off, but not on the test strip with the 200 ng/ml cut-off, thediazepam concentration in the filtrate can be reported as being in therange between 200 and 300 ng/ml.

Thus, through suitable selection and combinations of test strips,semiquantitative estimation of the diazepam concentration in thefiltrate is possible, thereby allowing determination of the relativealbumin-binding function.

What is claimed is:
 1. A method for determining an amount of functionalalbumin, the method comprising: a) providing a test sample containing adefined amount of albumin of unknown binding capacity and a referencesample containing the same defined amount of albumin having a referencebinding capacity; b) incubating the test sample and reference samplewith a defined amount of at least one albumin-binding marker M underconditions that allow the at least one albumin-binding marker M to bindto albumin (A) to form complexes of the at least one albumin-bindingmarker M and albumin (M:A); c) removing the complexes (M:A) formed instep c); d) detecting a presence or an amount of unbound marker M in thesamples after removal of the complexes (M:A) through a first and asecond test strips that allow for a determination of an amount ofunbound marker M, wherein the first and the second test strips havedifferent detection limits/tolerance limits for the marker M; and e)determining the amount of functional albumin based on the presence orthe amounts detected of marker M in step d).
 2. The method of claim 1,wherein the at least one albumin-binding marker M binds to binding siteI and/or II of albumin.
 3. The method of claim 1, wherein the at leastone albumin-binding marker M is a benzodiazepine.
 4. The method of claim3, wherein the benzodiazepine is diazepam.
 5. The method of claim 1,wherein the first and the second test strips has a predetermineddetection limit and/or a predetermined tolerance limit for the at leastone albumin-binding marker M.
 6. The method as claimed in claim 5,wherein the predetermined detection limit for the albumin-binding markerM is at least 100 ng/ml.
 7. The method as claimed in claim 5, whereinthe predetermined tolerance limit is about 200 ng/ml.
 8. The method ofclaim 5, wherein the first test strip has a predetermined tolerancelimit of about 200 ng/ml.
 9. The method of claim 5, wherein the secondtest strip has a predetermined tolerance limit of about 300 ng/ml. 10.The method of claim 1, wherein a plurality of test strips is used fordifferent albumin-binding markers M.
 11. The method of claim 1, whereinthe test sample is a sample from a patient with liver damage and/orrenal insufficiency and/or sepsis.
 12. The method of claim 1, whereinthe test sample is an albumin-containing solution.
 13. The method ofclaim 1, wherein the reference sample is a sample from a healthy subjector a synthetically produced albumin solution.